Complete Deracemization of Proteinogenic Glutamic Acid Using

Oct 18, 2012 - ABSTRACT: Viedma ripening is a process in which a compound that forms a stable racemic conglomerate can be converted to an...
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Complete Deracemization of Proteinogenic Glutamic Acid Using Viedma Ripening on a Metastable Conglomerate Laura Spix,† Hugo Meekes,† Richard H. Blaauw,‡ Willem J.P. van Enckevort,† and Elias Vlieg*,† †

Radboud University Nijmegen, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands Chiralix B.V., Toernooiveld 100, 6525 EC Nijmegen, the Netherlands

‡

S Supporting Information *

ABSTRACT: Viedma ripening is a process in which a compound that forms a stable racemic conglomerate can be converted to an enantiomerically pure solid state. Combining this deracemization process with Ostwald’s rule of stages, allows Viedma ripening to be extended to a racemic compound for which the conglomerate exists only in a metastable form. This is demonstrated for glutamic acid, a proteinogenic amino acid. Since Ostwald ripening is one of the processes occurring during Viedma ripening, we thus make use of Ostwald twice.

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acid in the world13 and the most abundant chiral, proteinogenic amino acid present in the Murchison meteorite.14 It is remarkable that all the amino acids found in this meteorite show an excess in the S enantiomer. In the case of glutamic acid it is between 50% and 70% ee depending on the extraction method.14 For glutamic acid the conglomerate is not the only reported crystal structure. Commercial (RS)-glutamic acid is a racemic hydrate.15,16 It exists also in the form of an anhydrous racemic compound.17 According to Menozzi,12 recrystallization from water is sufficient to get the glutamic acid conglomerate, but we could not reproduce this. After trying several recrystallization methods from acid solvents, it turned out to be possible to obtain the conglomerate by grinding the racemic hydrate in acetic acid at elevated temperatures (40−100 °C).

INTRODUCTION Several examples of complete deracemization of chiral solids using abrasive grinding have been reported in recent years. The first experiment was that of Viedma on NaClO3, a compound that is achiral in solution.1 Inspired by Viedma’s result, Noorduin et al.2 successfully adapted the method for an intrinsically chiral amino acid derivative that undergoes basecatalyzed racemization in solution. The method is now called Viedma ripening.3 In short, Viedma ripening involves the conversion of a racemic or scalemic mixture of a chiral compound into a single enantiomer by intense grinding of a slurry of the solids in a saturated solution, combined with a racemization process in the solution. A prerequisite of the process is that the compound forms a racemic conglomerate i.e. separate left and right handed crystals. In previous work, the possible relevance of Viedma ripening for the evolution of biomolecules to single handedness has been discussed.4−6 The first deracemization of a proteinogenic amino acid exploited an artificial conglomerate of aspartic acid obtained by mixing R and S solids.7,8 This compound forms a racemic compound under normal circumstances.9,10 Only 3 of the 20 proteinogenic amino acids are reported to form a racemic conglomerate (asparagine, threonine, and glutamic acid).10 Deracemization of natural amino acids using grinding, therefore, is quite a challenge. On top of that, even the mildest conditions for the necessary racemization still are rather harsh (100 °C in acetic acid with salicylaldehyde).11 Here we report the complete deracemization of a scalemic mixture of glutamic acid using Viedma ripening. Glutamic acid is one of the natural amino acids that is claimed to form a stable racemic conglomerate at room temperature.10−12 It is the industrially most produced amino © 2012 American Chemical Society

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EXPERIMENTAL SECTION

For the deracemization a suspension of glutamic acid (300 mg) with various initial enantiomeric excess (ee) values was stirred (260 rpm) in acetic acid (5 mL) at 70 °C in the presence of 2 mm glass beads (1.7 g). After the hydrate was converted into the conglomerate, as checked using X-ray powder diffraction (XRPD), the solution-phase racemization was initiated by adding salicylaldehyde (70 mg) and increasing the temperature to 90 °C. Samples of the solids were collected over time and the enantiomeric purity was measured using two independent chiral HPLC methods (see Supporting Information). Received: September 14, 2012 Revised: October 16, 2012 Published: October 18, 2012 5796

dx.doi.org/10.1021/cg301343a | Cryst. Growth Des. 2012, 12, 5796−5799

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Scheme 1. Equilibria Involved in the Attrition-Enhanced Deracemization of Glutamic Acid (Glu)

Figure 1. Attrition-enhanced evolution of solid-phase ee for glutamic acid; (a) at 90 °C starting from initial ee values as shown. (b) deracemization attempts at different temperatures all starting with ee = 64%. Lines are a guide to the eye.

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compound, following Ostwald’s rule of stages.23 This leads to a decrease in ee as the racemic compound cannot be deracemized and the racemization process will result in a zero ee in the end. To check this possibility, we examined the stability of the various crystal structures of glutamic acid using differentialscanning calorimetry (DSC) and XRPD (see Supporting Information). DSC indeed shows that the conglomerate is less stable than the anhydrous racemic compound, at least at temperatures near the melting point. XRPD confirms that under grinding conditions the racemic compound occurs after 20 h. The transition is completed after approximately 48 h. The hydrate is the least stable and does not play a role in the evolution of the solid phase ee. The relative stability at room temperature was determined by gently stirring of a mixture of crystals of two different glutamic acid structures in a saturated water or acetic acid solution. A mixture of the anhydrous racemic compound and the hydrate, which is also a racemic compound when stirred in water, evolved after two days into the pure anhydrous racemic compound, as shown using XRPD. A mixture of the conglomerate and the anhydrous racemic compound when stirred in acetic acid at room temperature, even after a week, still showed a mixture of these two compounds. Only after 30 days, it became apparent that the anhydrous racemic compound of glutamic acid is the stable one under these circumstances, although there were still traces of the conglomerate left. The

RESULTS AND DISCUSSION We observed that the continuous attrition of the crystals with an initial ee combined with the racemization in solution leads to a rise in ee in time. In contrast to the attrition enhanced deracemization experiments of Noorduin et al., however, in initial experiments the pure enantiomeric state was not reached by just prolonging the duration of the experiment (not shown). The experimental conditions were therefore adapted, to those described in the experimental section, such as to obtain complete deracemization within 24 h. The adaption included working with a higher starting ee, a rather large amount of glass beads and a reduced amount of glutamic acid to increase the deracemization rate.18 The results for the R and S enantiomer are almost identical (Figure 1a). The deracemization curves show the tendency to sigmoidal shapes (Figure 1a) as found in most of the experimental work1−3,5,7,18 and as explained in computer simulations of the process.19−22 Experiments with a low initial ee, however, showed a leveling off of the ee in time. (Figure 1a) Surprisingly, prolonging of the deracemization leads to a decrease of the solid phase ee even after having reached 100% (Figure 1b). Experiments which were conducted at lower temperature remained much longer at 100% ee (Figure 1b). A possible explanation for this is that the racemic conglomerate is metastable and is converted into the stable anhydrous racemic 5797

dx.doi.org/10.1021/cg301343a | Cryst. Growth Des. 2012, 12, 5796−5799

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Figure 2. Viedma ripening of glutamic acid at different temperatures and low starting ee values.

Scheme 2. Compounds and Crystal Structures That Participate in the Deracemization Processa

a

At high T, deracemization is hampered due to formation of racemic compounds.

formation of the glutamic acid racemic compound structure. To test this a Viedma ripening experiment was performed with and without pyroglutamic acid added at the start of the experiment. These experiments gave almost identical deracemization curves, so we conclude that the effect of pyroglutamic acid can be neglected (see Supporting Information). The experiments conducted at a lower temperature were more time-consuming. Nevertheless, they gave a better result in terms of the preservation of the conglomerate crystal structure. A complete deracemization with an initial ee of 14% was even possible (Figure 2a). Beginning at even lower initial ee’s displayed a definite increase in the solid phase ee (Figure 2b). Partial dissolving of a chiral scalemic compound leads to a higher ee in the solid phase. The same can happen if the compound decomposes. To distinguish between a rising ee because of decomposition or to Viedma ripening the yield of the grinding experiment must be determined. For glutamic acid, the yield is around 80% (see Supporting Information). No decomposition takes place during this experiment. The loss is due to the solubility of glutamic acid in the solvent and the

conversion of the conglomerate into the racemic compound in acetic acid is enhanced at elevated temperatures. This was proven by an experiment conducted at 40 °C in which the crystal structure was completely converted after 14 days. These results confirm the presumption that the conglomerate is kinetically formed according to Ostwald’s rule of stages and therefore metastable. This was already indicated in crystallization experiments of Viedma.24 The successful deracemization experiments were conducted at temperatures where the conglomerate survived long enough to reach 100% ee, before significant formation of the racemic compound. The racemization conditions for amino acids in acetic acid as solvent are typically 100 °C using salicylaldehyde as a catalyst. Figure 1b shows that increasing the temperature leads to a faster deracemization, probably partly due to a faster racemization in the liquid. On the other hand, the high temperature experiments result in a faster conversion into the racemic compound. Another effect that might play a role in the temperature dependent behavior is the formation of pyroglutamic acid in the solution which exists only as a racemic compound and might act as a kind of catalyst for the 5798

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(5) Noorduin, W. L.; Bode, A. A. C.; van der Meijden, M.; Meekes, H.; van Etteger, A. F.; van Enckevort, W. J. P.; Christianen, P. C. M.; Kaptein, B.; Kellogg, R. M.; Rasing, T.; Vlieg, E. Nature 2009, 1 (9), 729−732. (6) Crusats, J.; Veintemillas-Verdaguer, S.; Ribo, J. M. Chem.Eur. J. 2006, 12 (30), 7776−7781. (7) Viedma, C.; Ortiz, J. E.; de Torres, T.; Izumi, T.; Blackmond, D. G. J. Am. Chem. Soc. 2008, 130 (46), 15274−15275. (8) For amino acids, the R configuration corresponds in almost all cases to the D configuration, and S corresponds to L. (9) Flaig, R.; Koritsanszky, T.; Zobel, D.; Luger, P. J. Am. Chem. Soc. 1998, 120 (10), 2227−2238. (10) Jean Jacques, A. C.; Wilen, S. H. Enantiomers, Racemates, and Resolutions; Krieger: Malabar, FL, 1994. (11) Yamada, S.; Hongo, C.; Yoshioka, R.; Chibata, I. J. Org. Chem. 1983, 48 (6), 843−846. (12) A. Menozzi, G. A. Chem. Centralbl. 1894, 65, 463−464. (13) Izumi, Y.; Chibata, I.; Itoh, T. Angew. Chem., Int. Ed. 1978, 17 (3), 176−183. (14) Engel, M. H.; Nagy, B. Nature 1982, 296 (5860), 837−840. (15) Ciunik, Z.; Glowiak, T. Acta Crystallogr., Sect. C 1983, 39, 1271− 1273. (16) Flaig, R.; Koritsanszky, T.; Dittrich, B.; Wagner, A.; Luger, P. J. Am. Chem. Soc. 2002, 124 (13), 3407−3417. (17) Dunitz, J. D.; Schweizer, W. B. Acta Crystallogr., Sect. C 1995, 51, 1377−1379. (18) Noorduin, W. L.; Meekes, H.; van Enckevort, W. J. P.; Millemaggi, A.; Leeman, M.; Kaptein, B.; Kellogg, R. M.; Vlieg, E. Angew. Chem., Int. Ed. 2008, 47 (34), 6445−6447. (19) Noorduin, W. L.; Meekes, H.; Bode, A. A. C.; van Enckevort, W. J. P.; Kaptein, B.; Kellogg, R. M.; Vlieg, E. Cryst. Growth Des. 2008, 8 (5), 1675−1681. (20) Uwaha, M. J. Cryst. Growth 2011, 318 (1), 89−92. (21) Saito, Y.; Hyuga, H. J. Cryst. Growth 2011, 318 (1), 93−98. (22) Iggland, M.; Mazzotti, M. Cryst. Growth Des. 2011, 11 (10), 4611−4622. (23) Ostwald, W. Z. Phys. Chem. 1897, 22, 289−330. (24) Viedma, C. Origins Life Evol. Biospheres 2001, 31 (6), 501−509. (25) Ostwald, W. Lehrbuch der Allgemeinen Chemie; Verlag Von Wilhelm Engelmann: Leipzig, Germany, 1896; Vol. 2, Part1. (26) Liesegang, R. E. J. Z. Phys. Chem. 1911, 75, 374.

formation of a minor amount of pyroglutamic acid, mainly in the solution. We have thus found that the deracemization of glutamic acid in acetic acid is affected by several processes (scheme 2). In the beginning, just as in the experiments conducted by Noorduin et al., the deracemization rate constant is simply determined by the racemization efficiency via the solution.18 During the course of the experiment the formation of the racemic compound poses the largest problem, while the formation of pyroglutamic acid also becomes an issue, especially at the highest temperatures leading to a lower yield. These simultaneous processes, which occur all at different rates, act in a delicate balance and might explain the absence of the typical sigmoidal curves. (Figure 2a) The emergence of a solid phase with single chirality is therefore not guaranteed although still achievable by carefully tuning the conditions to minimize the side processes.

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CONCLUSION We have demonstrated a Viedma ripening method for the proteinogenic amino acid glutamic acid at temperatures far below literature suggestions.7,11 In the process, we make use of Ostwald twice. First of all Ostwald’s rule of stages,23 leading to the kinetic formation of the required conglomerate and second Ostwald ripening which plays an important role in Viedma ripening.3,25,26 It is the first example of a successful deracemization experiment employing a metastable conglomerate. This expands the application of Viedma ripening to substances which form a kinetic conglomerate.

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ASSOCIATED CONTENT

S Supporting Information *

Additional explicit information over all conducted experiments and additional figures showing XRPD measurements, DSC measurements, and comparison of two grinding experiments with and without additional pyroglutamic acid. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The work is part of the CW Programme Grant on “Chiral purification of racemic compounds: a grinding approach” and was made possible by financial support from the Netherlands Organization for Scientific Research (Nederlands Organisatie voor Wetenschappelijk Onderzoek (NWO)) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge Prof. Dr. R.M. Kellogg, Dr. B. Kaptein, and Dr. W.L. Noorduin for stimulating discussions. REFERENCES

(1) Viedma, C. Phys. Rev. Lett. 2005, 94 (6), 065504. (2) Noorduin, W. L.; Izumi, T.; Millemaggi, A.; Leeman, M.; Meekes, H.; Van Enckevort, W. J. P.; Kellogg, R. M.; Kaptein, B.; Vlieg, E.; Blackmond, D. G. J. Am. Chem. Soc. 2008, 130 (4), 1158−1159. (3) Noorduin, W. L.; van Enckevort, W. J. P.; Meekes, H.; Kaptein, B.; Kellogg, R. M.; Tully, J. C.; McBride, J. M.; Vlieg, E. Angew. Chem., Int. Ed. 2010, 49 (45), 8435−8438. (4) Viedma, C. Astrobiology 2007, 7 (2), 312−319. 5799

dx.doi.org/10.1021/cg301343a | Cryst. Growth Des. 2012, 12, 5796−5799